Preferential Recruitment of Th17 Cells to Cervical Cancer via CCR6-CCL20 Pathway

<div><p>Our previous studies suggest that Th17 cells accumulate within tumor tissues and correlate with recurrence of cervical cancer patients. However, the source of the increased tumor-infiltrating Th17 cells remains poorly understood. We investigated the prevalence, phenotype and trafficking property of Th17 cells in patients with cervical cancer. Our results showed that Th17 cells highly aggregated within tumor tissues in an activated phenotype with markedly increased expression of CCR6. Correspondingly, level of CCL20 in the tumor tissues was significantly higher than that in non-tumor and normal control tissues, and strongly positively associated with Th17 cells. Further, in vitro migration assay showed CCL20 had effective chemotaxis to circulating Th17 cells. In conclusion, Th17 cells are recruited into tumor tissues preferentially through CCR6-CCL20 pathway, which can serve as a novel therapeutic target for cervical cancer.</p></div

Association of intratumoral Th17-cell prevalence with clinical parameters.

<p>Association of intratumoral Th17-cell prevalence with clinical parameters.</p

Poly I:C induces IL-7 expression in the submandibular glands.

<p>(A) Real-time PCR analysis of gene expression in submandibular glands from C57BL/6 mice 6 hours post poly I:C injection, presented relative to that of β-actin. Data are the average of analyses of 6 individual mice (3 mice per experiment, total 2 independent experiments). (B) Immunofluorescence staining of IL-7 in submandibular gland sections from C57BL/6 mice 24 hourse post poly I:C injection. Differential interference contrast (DIC) image of the same sample is also shown. Data are representative of analyses of 6 individual mice (3 mice per experiment, total 2 independent experiments). (C) C57BL/6 mice were pretreated with anti-IFNAR1 or anti-IFN-γ 2 hours prior to poly I:C injection. After 6 hours, relative IL-7 mRNA levels in lung tissue were measured by real-time RT-PCR. Data are from analyses of 6 individual mice (2 mice per experiment, total 3 independent experiments). </p

Poly I:C induces production of IL-7 in human salivary gland epithelial cells.

<p>(A) HSG cells were cultured <i>in </i><i>vitro</i> with or without poly I:C for 1 and 3 days. IL-7 mRNA levels, left panel, and protein concentrations in culture supernatants, right panel, assessed by real time PCR and ELISA, respectively. (B) HSG cells were treated with IFN-α, IFN-γ or both for 1 and 3 days, and then measured for IL-7 mRNA levels. (C) HSG cells were treated with poly I:C in the presence of control IgG or anti-human IL-7 for 1 or 3 days, and then measured for BAFF mRNA expression. All PCR results are presented relative to that of GAPDH. All data are the average of analyses of 6 independent samples for each group (3 samples per experiment, total 2 independent experiments). </p

Poly I:C upregulates expression of CXCR3 ligands in submandibular glands in an IL-7-dependent fashion

<p>C57BL/6 mice were pre-treated with anti-IL-7Rα 2 hours prior to poly I:C injection. (A) Levels of CXCL9, 10, 11 and CXCR3 mRNA were measured from submandibular glands 24 hours after poly I:C administration, presented relative to that of β-actin. (B) Submandibular gland sections stained with CXCL9 and DRAQ5. All data are the average of or representative of 6 individual mice (2 mice per experiment, total 3 independent experiments).</p

Chemokine effects on Th17 cell recruitment.

<p>Migration assays were performed in a Transwell system. (A) Th17 cells migrate in response to recombinant human CCL17, CCL20 and CCL22 in dose-dependent manner (n = 5). Specific antibodies to chemokines significantly inhibit Th17 cell migration.***<i>P</i> < 0.001. (B and C) Expression of CCL20 by HeLa, Siha, and C-33A cells detected using Real-time PRC (B) and ELISA (C). All the three cervical cells could highly secrete CCL20 with highest level of CCL20 by HeLa cells. (D) Th17 cells also migrate toward culture supernatants of HeLa, Siha, and C-33A cells, which can be efficiently blocked by antibody against CCL20 alone or in combination with CCL17 and CCL22, but significantly less effectively by antibody against CCL17 or CCL22 (n = 3). *<i>P</i> < 0.05, **<i>P</i> < 0.01,***<i>P</i> < 0.001.</p

Inserting Pre-analytical Chromatographic Priming Runs Significantly Improves Targeted Pathway Proteomics with Sample Multiplexing

GoDig, a platform for targeted pathway proteomics without the need for manual assay scheduling or synthetic standards, is a powerful, flexible, and easy-to-use method that uses tandem mass tags to increase sample throughput up to 18-fold relative to label-free methods. Though the protein-level success rates of GoDig are high, the peptide-level success rates are more limited, hampering assays of harder-to-quantify proteins and site-specific phenomena. To guide the optimization of GoDig assays as well as improvements to the GoDig platform, we created GoDigViewer, a new stand-alone software that provides detailed visualizations of GoDig runs. GoDigViewer guided the implementation of “priming runs,” an acquisition mode with significantly higher success rates. In this mode, two or more chromatographic priming runs are automatically performed to improve the accuracy and precision of target elution orders, followed by analytical runs which quantify targets. Using priming runs, success rates exceeded 97% for a list of 400 peptide targets and 95% for a list of 200 targets that are usually not quantified using untargeted mass spectrometry. We used priming runs to establish a quantitative assay of 125 macroautophagy proteins that had a >95% success rate and revealed differences in macroautophagy expression profiles across four human cell lines

Phenotypic analysis of Th17 cells in patients with cervical cancer.

<p>(A) Representative expression profiles of CD45RO, HLA-DR, Granzyme B and PD-1 in tumor-infiltrating Th17 cells. The percentages represent the frequencies of various markers in Th17 cells. (B) Representative expression profiles of CCR4, CCR6 and CD49d on Th17 cells from peripheral blood (long dotted line), non-tumor (dotted line) and tumor tissues (solid line). The percentages represent the frequencies of various markers on tumor-infiltrating Th17 cells. (C) Statistical analysis of surface expression of CCR4, CCR6, CD49d on Th17 cells from peripheral blood, non-tumor and tumor tissues (n = 25). *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001.</p

NK cells contribute to the early up-regulation of CXCR3 ligands induced by poly I:C.

<p>(A) C57BL/6 mice were treated with poly I:C by for the indicated amount of time. Left panels, surface staining for immune cell populations in the submandibular glands, with the gating indicated above the plots. Right panels, Percentage of NK1.1⁺ TCR-β⁺, TCR-γδ⁺, CD4⁺ and CD8⁺ T cells among total submandibular gland cells. All data are the average of or representative of 5 individual mice (2-3 mice per experiment, total 2 independent experiments). (B) RAG-1^-/- mice were pre-treated with anti-NK1.1 antibody 2 days prior to poly I:C injection. Submandibular glands were harvested 6 hours after poly I:C injection and measured for gene expression by real time PCR. Data are from analyses of 4 individual mice (2 mice per experiment, total 2 independent experiments). </p

Poly I:C enhances Th1 and Tc1 responses in an IL-7-dependent manner.

<p>B6.NOD-<i>Aec</i> mice were injected with poly I:C plus IgG or anti-IL-7Rα antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077605#pone-0077605-g004" target="_blank">Figure 4</a>. (A) IL-7 mRNA levels in the submandibular glands of non-poly I:C- or poly I:C-treated B6.NOD-<i>Aec</i> mice. (B) Flow cytometric analyses of leukocyte subpopulations among mononuclear cells in submandibular glands. (C) Percentage of IFN-γ⁺ T cells in total submandibular gland cells, splenocytes and dr LN cells based on flow cytometric analysis. Data are representative of or the average of the analyses of 7 individual mice (1-3 mice per experiment, total 4 independent experiments). </p